ABSTRACT
A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.
Subject(s)
Azospirillum brasilense/genetics , DNA, Bacterial/genetics , Genes, Bacterial , Nitrogen Fixation/genetics , Bacterial Proteins/genetics , Gram-Negative Aerobic Bacteria/genetics , Klebsiella pneumoniae/genetics , Nitrogenase/genetics , Sequence Homology, Nucleic AcidABSTRACT
The use of electrokinetic phenomena should prove to be a promising bioseparation technique especially as an application of bioprocessing under microgravity. Therefore, a bioseparation Research and Development programme involving several research teams has been under way for three years in France. Based upon the results of this fundamental research, a programme is now proposed for the development of an automated process chain making it possible to obtain biological macromolecules of high purity under microgravity. This Space Bio Separation programme received the Eureka label at the 6th Ministerial Eureka conference. The project will last six years. It will involve the cooperation of several scientific and industrial partners in France, in Spain and in Belgium. The programme includes the development and validation of three different bioseparation facilities for space.
Subject(s)
Biotechnology/methods , Electrophoresis/methods , International Cooperation , Space Flight/instrumentation , Weightlessness , Belgium , France , Industry , Proteins/physiology , Research Design , SpainABSTRACT
The complete nucleotide sequence of the glnA gene, encoding the glutamine synthetase subunit of Azospirillum brasilense Sp7, was established. This is the first Azospirillum gene sequenced. The gene encodes a 468 residue polypeptide of MW 51,917. The similarity coefficient (SAB) between the polypeptidic sequence of Azospirillum and Anabaena 7120, which is the only other glnA sequence available, is 58%. No significant homology with E. coli canonical and ntr promoters, or with the promoter region of the Anabaena glnA gene was found. When fused to an E. coli promoter, the gene could be translated in E. coli, despite a very biased codon usage and an atypical Shine-Dalgarno sequence.
Subject(s)
Genes , Glutamate-Ammonia Ligase/genetics , Gram-Negative Bacteria/enzymology , Amino Acid Sequence , Base Sequence , Codon , Cyanobacteria/enzymology , Escherichia coli/enzymology , Genes, Bacterial , Gram-Negative Bacteria/genetics , Promoter Regions, Genetic , Protein Biosynthesis , Transcription, GeneticABSTRACT
A plasmid which, by complementation, restored a Gln+Nif+ phenotype to the Gln-Nif- Azospirillum brasilense mutant 7029, was isolated from a gene bank of total DNA of A. brasilense Sp7 (ATCC 29145) constructed in the broad host range vector pVK100. This plasmid contained the structural gene (glnA) for glutamine synthetase. The glnA gene was mapped by Tn5 insertion and DNA hybridization with a Klebsiella pneumoniae glnA probe. The direction of transcription of glnA was determined. The glnA product was identified as a 50-Kd polypeptide which could be adenylylated in Escherichia coli, and glutamine synthetase activity was characterized in E. coli. Plasmids containing the glnA gene restored glutamine-independent growth and a Nif+ phenotype to Gln-Nif- and Gln-Nifc mutants of Azospirillum.
